Background: Roselle (Hibiscus sabdariffa L.) calyces have been used in traditional medicine as diuretics, mild laxatives, and antihypertensive agents but to date, a comprehensive study of its pharmacological activity and safety has not been conducted.

Aims of the study: The current study aims to provide a comprehensive evaluation of the antihypertensive efficacy and toxicity profile of Roselle (H. sabdariffa L.) calyces extract. Utilizing animal models, the investigation assessed the dose-dependent pharmacological effects and safety of H. sabdariffa L.

Results: The findings indicate that the extract exerts a significant antihypertensive effect at a dose of 250 mg/kg body weight (BW), lowering systolic and diastolic blood pressures by 10.12% and 11.63%, respectively. Ethyl acetate fractions administered at 112.5 mg/kg BW demonstrated greater efficacy than n-hexane and aqueous fractions, suggesting that the active compounds likely possess semi-polar properties. Acute toxicity testing yielded an LD₅₀ of 8.75 g/kg BW for male rats and 7.5 g/kg BW for female rats, classifying the extract as slightly toxic. The sub-chronic toxicity study shows that H. sabdariffa L. demonstrates an effect on bodyweight and urea levels in male and female rats, while the change in the blood parameters, creatinine level, and the liver index was only observed in female rats. Conclusions: These data suggest that H. sabdariffa L. extract exhibits therapeutic promise but should be administered cautiously, preferably at doses lower than 250 mg/kg BW, due to potential toxicity.

acute, diastolic, sub-chronic, systolic, toxicity

Hypertension is a risk factor for cardiovascular disease which has a high prevalence and mortality (Pattanittum et al. 2010; Stanaway et al. 2018). It is defined as a systolic blood pressure of 140 mmHg or higher and/or diastolic blood pressure of 90 mmHg or higher (Unger et al. 2020). There are various pharmacological approaches to lowering systolic and diastolic blood pressures in adults, such as the administration of antihypertensive drugs such as beta-blockers, thiazide, and thiazide-like diuretics, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, and calcium channel blockers (Pattanittum et al. 2010; Laurent 2017; Williams et al. 2018). However, the use of such classes of antihypertensive drugs is associated with undesirable side effects such as hypokalemia, nausea, dizziness, and dry cough, thus there is a need for alternative medicine (Hopkins et al. 2013; Laurent 2017; Jeffery and Richardson 2021; Salem et al. 2022).

Medicinal plants are of great interest because they are an abundant natural source of novel therapeutic agents for the treatment and prevention of hypertension (Jacob and Narendhirakannan 2019; Ben El Mostafa and Maamri 2020; Jadid et al. 2020; Jeffery and Richardson 2021). For example, Roselle (Hibiscus sabdariffa, Malvaceae family) is a medicinal plant which has been widely used to treat hypertension, particularly in Malaysia and Indonesia where it is consumed as a refreshing drink (Pattanittum et al. 2010; Carvajal-Zarrabal et al. 2012; Hopkins et al. 2013; Ali and El-Anany 2017).

Several animal studies have demonstrated that H. sabdariffa has several pharmacological activities including antihypertensive, antidiabetic, antibacterial, antioxidant, anticholesterol, and hepatoprotective properties (Aba et al. 2014; Adeyemi et al. 2014; Asgary et al. 2016; Ali and El-Anany 2017; Mohammed Yusof et al. 2017; Adusei 2020; Jeffery and Richardson 2021; Salem et al. 2022). The antioxidant properties of roselle are associated with its anthocyanin content and protect cells from free radical damage caused by exposure to UV light (Nurkhasanah et al. 2016; Rizki et al. 2017; Adusei 2020).

The main bioactive constituents responsible for the physiological activities of H. sabdariffa calyces are organic acids, mainly citric and malic acids, anthocyanins, a myriad of flavonoids and glycosides, and fibre (Carvajal-Zarrabal et al. 2012; Hopkins et al. 2013). A recent phytochemical study of H. sabdariffa revealed the presence of phenolic components, anthocyanins, flavonols, and protocatechuic acid (PCA) (Essa et al. 2006). The pharmacological activity of roselle is attributed to the presence of gossypetin, hibiscine glucoside, and anthocyanins. Delphinidin 3-sambubioside and cyanidin 3-sambubioside are two anthocyanin compounds that are generally believed to be the active constituents responsible for the antihypertensive, antioxidant, and hypocholesterolemic effects of H. sabdariffa (Hopkins et al. 2013). Additionally, calyces of H. sabdariffa contain several amino acids that are very important for improving body health (Da-Costa-Rocha et al. 2014; Jeffery and Richardson 2021).

There are various studies regarding the pharmacological potential of roselle extract, however, a comprehensive study of the antihypertensive effects of roselle calyces extract and active fractions, as well as its safety profiles is limited. Therefore, this study aims to complement and update the information reported previously in the literature.

Hibiscus sabdariffa L. (Malvaceae) tea is widely consumed as a beverage and as a treatment for hypertension and hyperlipidemia (Hopkins et al. 2013). Roselle drink has been reported could elevate plasma high‑density lipoproteins, and significantly reduced both systolic and diastolic blood pressure in healthy subjects (Diantini et al. 2021). Moreover, a recent clinical trial reported that 6 months of administration of roselle drink, could improve the levels of glutathione, superoxide dismutase (SOD), glucose-6-phosphate dehydrogenase (G-6-PDH), glutathione peroxidase (GSH-Px), and glutathione reductase (GSH-Rd) in healthy subjects (Chiu et al. 2022). In traditional medicine, either maceration with cold water or hot decoction is the most widely applied extraction method for the preparation of roselle herbal teas (Salem et al. 2022). In their study, Hopkins (2013) concluded that there are consistent results on the beneficial effects of HS extracts on lowering blood pressure. Although the same effects on blood pressure occur in normotensive animal models, the positive effects are greater in hypertensive animals. There is evidence that the positive effects are dose-dependent at lower doses.

Administration HS extract and fractions markedly reduced the elevated blood pressure that could be related to the anthocyanins’ hypotensive constituent of Hibiscus sabdariffa, including delphinidin-3-O-sambubioside (hibiscin) and cyanidin-3-O-sambubioside (gossypicyanin), which were reported to possess the ACE inhibition activity (Salem et al. 2022).

In our study, the ethanol extract and ethyl acetate fraction showed significant antihypertensive effects and were known to contain secondary metabolites of the flavonoid and polyphenol groups. This was also reported by other studies which stated that the main bioactive compounds in roselle flower petals were flavonoids, polyphenols and anthocyanins, as well as organic acids (Da-Costa-Rocha et al. 2014).

Medicinal plants are presumed to be safe without any compromising health effects. Appropriate use of medicinal plants in dietary supplementation is very important in the maintenance of health (Manaharan et al. 2014). Many studies have reported the harmful effects of the improper use of medicinal plants. Therefore, evaluating the toxicological effects of any medicinal plant extract intended to be used in animals or humans is a crucial part of its assessment for potential toxic effects. This study finds that H. sabdariffa belongs to the slightly toxic category because the dose is in the 5–15 g/kg BW.

Different from several previous similar studies, Sari et al. (2016) reported that the administration of ethanol extract of rosella calyces to SD rats found the LD₅₀ at 850.90 mg/kg BW. Adeyemi et al. (2014) reported that methanol extract of roselle calyces also showed LD₅₀ at a dose of 3200 mg/kg BW within 72 hours. Sireetawong et al. (2013) reported that water extract with a single dose 5,000 mg/kg BW did not cause death in test animals for 14 days (Sireeratawong et al. 2013; Adeyemi et al. 2014; Sari et al. (2016)).

While sub-chronic toxicity deals with the adverse effects of single doses, many chemical substances which are given in repeated doses do not produce immediate toxic effects. Delayed effects may occur due to the accumulation of the chemical in tissues or to other mechanisms. Thus, it is important to identify any toxic potential of the substance by a sub-chronic toxicity test.

The use of a satellite group of test animals, given the highest dose and then observed after the ending of dosing, is to give additional information on the persistence or reversibility of effects (Sireeratawong et al. 2013).

According to Manaharan et al. (2014), after some exposure to potentially toxic substances, there will be a slight reduction in body weight gain. Our study finds that the average body weight of male and female rats from each group was significantly different, which in the test group was lower than in the control group. This suggests that administration of the HS extract affects the body weight of the rats. We also observed, there was a significant difference in the liver organ index in female rats from the satellite group to other groups, while the kidney organ index had no significant difference.

The gross and microscopic observations conducted in all the above-mentioned organs further suggested there is minor damage caused by the administration of the extract at the concentrations studied for 90 days consecutively.

The previous reports on the toxicity profile of HS showed no observed toxicity at 15 g/kg calyces (high dose) of aqueous and ethanol extracts in mice within 7 days after oral administration. Sireeratawong et al. (2013) also reported the safety HS (red species) aqueous calyces extract when administered a single oral dose of 5,000 mg/kg BW after fourteen days. They also reported safety at doses between 50 and 200 mg/kg BW of extract after oral administration every day for two hundred and seventy days.

This study revealed H. sabdariffa extract has some toxic effects in rats on sub-chronic administration. In addition, the extracts produced a significant diuretic activity. Hence, prolonged oral consumption of the extract may not be recommended (Njinga et al. 2020).

Based on the comprehensive analysis, it is evident that the ethyl acetate fraction of Hibiscus sabdariffa L. petals exhibits remarkable antihypertensive activity comparable to that of the positive control. In the context of acute toxicity, the ethanolic extract from roselle calyces falls within the classification of slight toxicity. Neuropharmacological assessment indicates that the test preparation has a profound impact on both central and autonomic nervous system activities. Sub-chronic toxicological evaluations reveal significant adverse effects in specific physiological parameters for both male and female Wistar rats, in addition to minor hepatic and renal damage. Therefore, caution is advised when administering the extract, particularly at doses approaching or exceeding the identified threshold, underscoring the necessity for further mechanistic studies and clinical evaluations to substantiate the safety and efficacy of this plant-based intervention.

The authors greatly acknowledge the financial support from The Ministry of Research and Technology of Higher Education, PUPT Scheme, Grant No. 1827/UN6.3.1/LT/2020. The APC was fully funded by Universitas Padjadjaran via the Directorate of Research and Community Engagement. We would also like to thank Prof. Dr. Apt. Jutti Levita for her valuable advice and the research team of the Center of Herbal Studies: Santi Perawati, Annisa Nur U. P., Tanty Novianti Riani, and Khaerunnisa Sekar Ningrum who assisted the technical testing of pharmacology and compiled the research report.